Affinity labeling of residues within Hv2 of guinea pig anti-azobenzenearsonate antibodies of different isotypes and from different strains.
Anti-p-azobenzenearsonate (ARS) antibodies of IgG1 and IgG2 isotypes produced in inbred strain 13 and strain 2 guinea pigs were affinity labeled with N-(bromoacetyl)-3-[(p-arsonophenyl)azo]-L-tyrosine (BAAT) or N-(bromoacetyl)-p-arsanilic acid (BAA). BAAT was shown to modify approximately 50% of the binding sites specifically and BAA approximately 30%. Both reagents preferentially modified residues in the heavy (H) chain to the extent that it contained over 80% of the affinity label associated with the native molecule. At least 80% of label borne by the variable domain of the H chain (VH) was found in the second hypervariable region (Hv2). BAAT labeled all anti-ARS antibodies exclusively at position N-59, which contains a lysyl residue. BAA labeled predominantly tyrosine at N-57 and, to a lesser extent, lysine-59 and tyrosine-50. Comparison of Hv2 sequences in anti-ARS and in antibodies reactive with other haptens has shown that tyrosine at N-50 and N-57 as well as lysine at N-59 is distinctive of antibodies with anti-ARS specificity, thus implying their involvement in antigen binding. The predominant sequence of Hv2 was identical in anti-ARS IgG1 and IgG2 molecules induced in either inbred guinea pig strain following either carrier priming or conventional immunization. Although limited variability does occur among the various populations of anti-ARS antibodies in certain residue positions in Hv2, no significant differences in the binding affinities or in the indexes of heterogeneity were seen among the various kinds of anti-ARS antibodies.
D000345:Affinity Labels; D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000032:analysis*; D000936:Antigen-Antibody Complex; D001147:Arsanilic Acid / Q000031:analogs & derivatives*; D001152:Arsenicals; D001391:Azo Compounds / Q000032:analysis*; D006168:Guinea Pigs; D007126:Immunoglobulin Allotypes; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains / Q000302:isolation & purification; D007147:Immunoglobulin Light Chains / Q000302:isolation & purification; D007135:Immunoglobulin Variable Region / Q000302:isolation & purification; D007700:Kinetics; D013045:Species Specificity; D010132:p-Azobenzenearsonate / Q000032:analysis*
D000345:Affinity Labels; D000888:Antibodies, Anti-Idiotypic; D000936:Antigen-Antibody Complex; D001152:Arsenicals; D001391:Azo Compounds; D007126:Immunoglobulin Allotypes; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; C030300:N-(bromoacetyl)-4-arsanilic acid; D010132:p-Azobenzenearsonate; C030301:N-(bromoacetyl)-3-((4-arsonophenyl)azo)tyrosine; D001147:Arsanilic Acid